Kit for radiolabelling with 68ga comprising a metal inhibitor

ABSTRACT

The present invention relates to a kit for radiolabelling a targeting agent with gallium-68. The present invention also relates to the use of said kit for radiolabelling a targeting agent, a method for radiolabelling a targeting agent with gallium-68 using said kit, said kit and a method of preparation.

TECHNICAL FIELD

The present invention is related to kit for radiolabelling BACKGROUND

Recently, some very interesting clinical results based on gallium-68radiolabeled molecules for imaging in vivo by PET were published andpresented. These radiotracers are generally made by assembly of achelating agent with a targeting agent, generally DOTA-functionalizedtargeting agents, allowing, respectively, the reaction with a metallicradioisotope or radiometal and biological/metabolic activity of theradiotracer. However, due to the short half-life of gallium-68 (68minutes), the radiotracer, i.e. radiolabelled chelate-functionalizedtargeting agent, based on this radioisotope are not suitable forlong-distance distribution and require on the spot production andsuitable production equipment, such as automated synthesizers, for theradiolabelling process, making it difficult for widespread use inroutine nuclear medicine.

The labelling reaction with the gallium-68 is performed by chelating theradioactive metal with a suitable chelating agent in a suitable reactionmedium, usually in a buffered medium in order to ensure an optimum pHfor both the chelation reaction and the gallium solubility.

Gallium-68 itself is obtained from a generator. Said generator is analternative to the in situ production using a cyclotron or dailydelivery of radioisotopes. The system was initially developed fortechnecium-99. The principle is based on the radiochemical separationbetween a parent element of long half-life (or nonradioactive elementssuch as germanium-68) contained in the generator and a daughter elementwhich is a short half-life element resulting from the disintegration ofthe parent element. The daughter is recovered with excellentradiochemical purity and radionuclidic properties (i.e. withoutcontamination from other radionuclides or other radiochemicalimpurities) and with good chemical purity (low metal ion content). Thisseparation is made possible by the different chemical properties of thetwo elements (parent and daughter).

The characteristics of a germanium-68/gallium-68 generator can besummarized as follows:

-   -   The eluate is obtained in an acid solution (0.05M-5M HCl,        specified by the manufacturer of the generator)    -   The eluate contains zinc-68, resulting from both the        manufacturing process of germanium-68 and disintegration of        gallium-68, whose concentration increases continuously in        function of time elapsed since the last elution of the        generator. Indeed, this zinc-68 accumulates in the generator.        This can be detrimental to the performance of radiolabelling        since this zinc-68 enters in direct competition with gallium-68        for chelation reactions used for radiolabelling.    -   The eluate further contains germanium-68 (the “breakthrough”)        released from the generator.    -   The eluate also contains a variety of metal leaching from the        solid phase of the generator column, tubings, but also brought        by the HCl used for elution:        -   Microg/ml level: Fe (III), Zn (II), Al (III)        -   Picog/L level: Mn (II), Pb (II), Ti (IV), Cr (III), Ni (II)            (Sn (IV))

The efficiency of the chelation reaction is dependent on a suitable pH,but also on possible competition of the metallic impurities mentionedabove with the gallium-68 during the chelation reaction as well. Inaddition, it is generally accepted that heat facilitates the chelationreaction for the most commonly used gallium-68 based radiotracers.

In the state of the art, the presence of metal ions that compete withgallium-68 is generally reduced by pre-labelling purification orfractionation of the eluate (as described in WO 2010/092114). Theseadditional steps however represent a loss of radioactivity resultingfrom, either wasted time or the process itself. These losses can reachup to 30% of the total radioactivity, respectively, 10% due to decay and20% coming from the pre-purification process itself.

The possibility of partial chelation of gallium-68 requires, in general,a final post-labelling purification in order to obtain a radiotracerhaving a radiochemical purity that meets the pharmaceuticalspecifications (>90% radiochemical purity). These steps also representan additional loss of activity that can rise to up to 10% resulting fromwasted time or the process itself.

According to known processes, at the end of the radiolabelling, asequestering agent having a particular affinity for the gallium-68 maybe added to chelate the non-reacted part of the isotope. This complexformed by the sequestering agent and the non-reacted gallium-68 is thendiscarded in order to reach a better radiochemical purity afterradiolabelling.

In addition, the need for these pre- and post-labelling purificationsteps makes these gallium-68 labeled radiotracer synthesis dependent, tosome extent, on automation and on the use of a synthesis module. Inaddition to technical expertise, this requires extra time lossunfavorable to the overall performance.

Due to the short half-life of the radionuclide (68 minutes) and tolimited activity supplied by the generator (max. 100mCi), anyimprovement in order to achieve rapid, direct and high efficiencychelation of target molecules is thus highly desirable.

In order to maintain the pH of the labelling solution in a range whereit is possible to ensure both the chelating reaction and the gallium-68solubility, a buffering medium is generally used. The desired buffermust be nontoxic, must effectively maintain the pH within a range of 3.0to 5.0, should not compete with gallium-68 ions and have preferably alow capacity for metal chelation with regard to the capacity of thechelating agent as assembled with the targeting agent. It must also beable to tolerate possible small changes in the volume of generatoreluate (and therefore the amount of HCl), i.e. it must be strong enoughto maintain the pH within the desired range with 10% changes in thevolume of eluate.

Management of competing metal impurities is another challenge. It hasbeen shown in WO2013024013 that adding a co-chelating agent could allowinhibition of competing metal impurities. Indeed, any species that wouldinhibit metal impurities by avoiding or having limited capacity tointerfere negatively on the gallium-68 chelation reaction can act as atrap for these impurities. In other words, this inhibitory effect bringsthe apparent concentration of competitor metal, i.e. the concentrationof metallic impurities yet available for chelation to a level whichallows high yields and reproducible radiolabelling. This co-chelatingagent is by definition different than the chelating agent assembled withthe targeting agent.

In this context, it is clear that a need exists for an improved processfor the preparation of ⁶⁸Ga complex which overcomes one or more of theabove mentioned problems. This involves identifying an appropriatemedium that maintains the pH within a tolerable range, to handle themetal contamination, which avoids the need to heat for promoting thechelating reaction and allows gallium-68 chelation yields upper 90%.

SUMMARY OF THE INVENTION

The present invention relates to the following aspects:

Aspect 1. A radiolabelling kit comprising:

-   -   a suitable amount of acetate salt or buffer to balance at least        the acidic pH eluate from a gallium-68 generator to a pH value        ranging from 3 to 5 when said generator is eluted in the kit;    -   a chelate-functionalized targeting agent, said chelate function        being able to chelate gallium-68 in the radiolabelling        conditions; and    -   a metal inhibitor, which is a co-chelating agent, capable of        inactivating contaminating metals other than gallium-68 without        interfering with the chelation between gallium-68 and said        chelate-functionalized targeting agent, under the conditions of        the labelling reaction.

Aspect 2. The kit according to aspect 1, wherein said acetate salt,chelate-functionalized targeting agent and metal inhibitor are(co-)lyophilized.

Aspect 3. The kit according to aspect 1, wherein saidchelate-functionalized targeting agent and metal inhibitor are(co-)lyophilized, and wherein said acetate salt or buffer is presentseparately and can be added subsequently.

Aspect 4. The kit according to aspect 1, wherein saidchelate-functionalized targeting agent and metal inhibitor are(co-)lyophilized, an acetate buffer being added subsequently.

Aspect 5. The kit according to anyone of aspects 1 to 4, wherein theacetate salt or acetate salt buffer comprises a quantity of acetate saltadjusted to the type of the gallium-68 generator used.

Aspect 6. The kit according to anyone of aspects 1 to 4, wherein theacetate salt or acetate salt buffer is present in a fixed concentrationto balance the acidic pH eluate from a gallium-68 generator to a pHvalue ranging from 3 to 5 when said generator is eluted in the kit afteraddition of HCL to the kit.

Aspect 7. The kit according to anyone of aspects 1 to 6, wherein thechelate functional group of the targeting agent is capable of forming astable complex with Ga3+.

Aspect 8. The kit according to anyone of aspects 1 to 7, wherein thechelate functional group of the targeting agent is selected from thegroup comprising: NOTA and derivatives, Tris(hydroxypyridinone) (THP)and derivatives, open-chain chelators such as HBED, MPO, EDTA, 6SS,B6SS, PLED, TAME, and YM103; NTP (PRHP) 3, H2dedpa and its derivatives,(4,6-MeO2sal) 2-BAPEN, and citrate and its derivatives.

Aspect 9. The kit according to anyone of aspects 1 to 8, wherein theacetate salt is present in an of amount between 5 mg and 1000 mg,preferably in an amount of between 10 mg and 750 mg, more preferably inan amount of between 20 mg and 500 mg.

Aspect 10. The kit according to anyone of aspects 1 to 9, wherein themetal inhibitor is present in a micromolar quantity, preferably in ananomolar quantity, more preferably in a quantity of below 500nanomoles, even more preferably in a quantity of below 100 nanomoles.

Aspect 11. The kit according to anyone of aspects 1 to 10, wherein saidmetal inhibitor is selected from the group comprising: DOTA and itsderivatives, DTPA and its derivatives, and sugars.

Aspect 12. The kit according to anyone of aspects 1 to 11, wherein saidmetal inhibitor is selected from the group comprising: monosaccharidesand their derivatives, disaccharides and their derivatives, andpolysaccharides and their derivatives.

Aspect 13. The kit according to anyone of aspects 1 to 12, wherein saidmetal inhibitor and said functionalised agent are chemically linked.

Aspect 14. The kit according to anyone of aspects 1 to 13, wherein saidmetal inhibitor and said functionalised agent are chemically linked,through a linker that is unstable in the radiolabelling conditions.

Aspect 15. Use of the kit according to anyone of aspects 1 to 14, forradiolabelling a chelate-functionalized targeting agent with gallium-68carried out at a temperature near or equal to room temperature.

Aspect 16. A method for radiolabelling a chelate-functionalizedtargeting agent with gallium-68, comprising the elution of a gallium-68generator with an eluent comprising an acid, in a kit according toanyone of aspects 1 to 13.

Aspect 17. The method according to aspect 16, wherein the acid is HCl.

Aspect 18. The method according to aspect 16 or 17, comprisingadditionally the step of adding HCl to the kit before elution.

Aspect 19. The method according to anyone of aspects 16 to 18, whereinthe radiolabelling is performed at a pH comprised between 3 and 5,preferably between 3,5 and 4,5, more preferably between 3,9 and 4,3.

Aspect 20. The method according to anyone of aspects 16 to 19, whereinthe radiolabeling reaction is carried out at a temperature of below 50°C., preferably of ambient or room temperature (e.g. of between 20 and30° C.).

Aspect 21. A solution obtainable by elution of a gallium-68 generatorwith an eluent comprising an acid, in a kit according to anyone ofaspects 1 to 14.

Aspect 22. A solution obtainable by elution of a gallium-68 generatorwith an eluent comprising a base, in a kit according to anyone ofaspects 1 to 14.

Aspect 23. A solution obtainable by elution of a gallium-68 generatorwith an eluent that is concentrated or purified prior to its transfer ina kit according to anyone of aspects 1 to 14.

Aspect 24. The solution according to anyone of aspects 21 to 23, havinga pH of between 3 and 5, preferably between 3,5 and 4,5, more preferablybetween 3,9 and 4,3.

Aspect 25. A process for preparing a radiolabelling kit according toanyone of aspects 1 to 14, comprising the steps of:

a) preparing or providing a solution comprising suitable amount ofacetate salt or buffer to balance at least the acidic pH eluate from agallium-68 generator to a pH value ranging from 3 to 5, preparing orproviding a chelate-functionalized targeting agent and preparing orproviding an inhibitor of metal; and

b) lyophilizing the solution obtained in step a).

Aspect 26. A process for preparing a radiolabelling kit according toanyone of aspects 1 to 14, comprising the steps of:

a) preparing or providing a solution comprising a chelate-functionalizedtargeting agent and an inhibitor of metal;

b) lyophilizing the solution obtained in step a), and

c) adding the acetate salt as a powder in the obtained lyophilizedproduct in step b).

Aspect 27. A process for preparing a kit according to anyone of aspects1 to 14, comprising the steps of:

a) preparing a solution comprising a chelate-functionalized targetingagent and an inhibitor of metal;

b) lyophilizing the solution obtained in step a), and

c) adding an acetate buffer in the obtained lyophilized product in stepb).

Aspect 28. The invention further provides a process for preparing a kitaccording to anyone of aspects 1 to 14, comprising the steps of:

a) preparing a solution comprising the acetate salt, achelate-functionalized targeting agent and an inhibitor of metal; and

b) optionally freeze the solution obtained in step a).

Aspect 29. A process for radiolabelling a target agent with gallium-68,wherein a metal inhibitor is included either in the eluate of thegallium-68 generator, or in the HCl solution added before elution of thegallium-68 generator, wherein said metal inhibitor is present in theradiolabelling solution.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. The terms also encompass“consisting of” and “consisting essentially of”.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of and from the specified value, inparticular variations of +/−10% or less, preferably +/−5% or less, morepreferably +/−1% or less, and still more preferably +/−0.1% or less ofand from the specified value, insofar such variations are appropriate toperform in the disclosed invention. It is to be understood that thevalue to which the modifier “about” refers is itself also specifically,and preferably, disclosed.

Whereas the term “one or more”, such as one or more members of a groupof members, is clear per se, by means of further exemplification, theterm encompasses inter alia a reference to any one of said members, orto any two or more of said members, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or≥7 etc. of said members, and up to all said members.

All documents cited in the present specification are hereby incorporatedby reference in their entirety.

Unless otherwise specified, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions may be includedto better appreciate the teaching of the present invention.

In the following passages, different aspects or embodiments of theinvention are defined in more detail. Every aspect or embodiment sodefined may be combined with each of the other aspects or embodimentsunless stated otherwise. In particular, any feature indicated as beingpreferred or advantageous in one embodiment may be combined with anyother embodiment or embodiments indicated as being preferred oradvantageous.

The present invention overcomes one or more of the problems identifiedand observed in the state of the art and allows the directradiolabelling of a chelate-functionalized targeting agent withgallium-68 at a temperature below 50° C. and preferably at roomtemperature, using a kit as described herein, this gallium-68 beingeluted from a germanium-68/gallium-68 generator in an acidic aqueoussolution.

Accordingly, in one aspect, the invention provides a kit comprising:

-   -   A suitable amount of acetate salt to balance at least the acidic        pH eluate from a gallium-68 generator to a pH value ranging from        3 to 5 when said generator is eluted in the kit; and    -   A chelate-functionalized targeting agent, able to chelate        gallium-68 in the radiolabeling conditions    -   A metal inhibitor, which is a co-chelating agent, capable of        inactivating metals other than gallium-68 without interfering        with the chelation between gallium-68 and the said        chelate-functionalized targeting agent, under the conditions of        the labelling reaction. In other words, said metal inhibitor is        selected for its ability to chelate contaminating metals        interfering and competing with the chelation of gallium-68 while        being mostly unable gallium-68 in the said conditions of the        labelling reaction as opposed to the chelate-functionalized        targeting agent.

Said kit being suitable to perform the radiolabelling reaction of saidchelate-functionalized targeting agent with gallium-68 as carried out ata temperature near or equal to room temperature, preferably at atemperature below 50° C. and more preferably at room temperature.

The invention also relates to a kit wherein the acetate salt, thechelate-functionalized targeting agent and the metal inhibitor are (co-)lyophilized.

The invention also relates to a kit wherein the chelate-functionalizedtargeting agent and the metal inhibitor are (co-)lyophilized, theacetate salt being added subsequently.

The invention also relates to a kit wherein the chelate-functionalizedtargeting agent and the metal inhibitor are (co-)lyophilized, an acetatebuffer being added subsequently.

The invention also relates to a kit wherein the acetate salt, thechelate-functionalized targeting agent and the metal inhibitor aresolubilized and further frozen.

The kit as described herein can not only provide an optimum pH forcarrying out the chelation reaction or radiolabelling, but also allowsto tolerate or manage the variation of the eluate volume and acidityassociated with different types of gallium-68 generators, through theuse of a suitable amount of acetate salt that when mixed with the acidgenerator eluate, form an acetic acid/acetate buffer having an acid pHcomprised in the interval 3-5. In these conditions, the amount ofnon-chelated gallium-68 because of a too low or too high pH, which leadsrespectively to a high content of free gallium-68 cations or togallium-68 hydroxides (gallium colloids), is minimized.

In addition, the acetate buffer is well tolerated as a buffer or as anexcipient for pharmaceuticals.

Furthermore, the present inventors have found that a metal inhibitor canbe used in the radiolabelling method for neutralizing, at leastpartially, interfering species and allows the gallium-68 to react withthe chelate-functionalized targeting agent. These metal inhibitors maytemporarily or permanently remove metals that compete with gallium-68for the reaction with the chelate-functionalized targeting agent. Saidmetal inhibitor is thus unable to chelate gallium-68 in the saidconditions of the labelling reaction, but chelate other metalsinterfering with the chelation of gallium-68 by thechelate-functionalized targeting agent. The presence of a metalinhibitor during the radiolabelling reaction provides an advantageousalternative to current approaches for managing the presence of metallicimpurities, such as increasing the amount chelate-functionalizedtargeting agent, or the pre-treatment of the eluate of the generator,since these additional purification steps consume time (andradioactivity).

These aspects as described herein advantageously allow obtaining anappropriate chelation yield, particularly of about 90% and more, andtherefore a sufficient radiochemical purity without any preliminary orfurther final purification.

The presence of a chelate-functionalized targeting agent, an acetatesalt and a metal inhibitor in the kit advantageously allows to directlyelute gallium-68 generator in the kit and performing the radiolabellingreaction without the need for any prior or subsequent operation.

In addition, all kit components as described herein can be lyophilizedaltogether or frozen which ensures a longer shelf life.

Thus, the main advantages of a kit as disclosed herein thatdifferentiate said kit from the state of the art are:

-   -   A completely dry or frozen kit that allows a better shelf life        of the chelate-functionalized targeting agent;    -   The possibility of radiolabelling without the need for an        automated synthesizer;    -   The possibility of a radiolabelling without the need for        heating;    -   The presence of a metal inhibitor which advantageously allows to        use less chelate-functionalized targeting agent and allowing the        implementation of more affordable radiopharmaceutical synthesis;    -   The presence of a metal inhibitor which advantageously allows to        improve the radiolabelling yields;    -   The fact that any brand generator can be used with this kit        provided as acetate or partially neutralized with HCl so that        when mixed with the acid generator eluate, the optimal pH for        the radiolabelling is obtained.

As used herein, “acetate” refers to the anionic molecule CH₃COO—. Theterm “acetate salt” herein is meant any metal salt acetate. Non-limitingexamples of acetate salts include sodium acetate, potassium acetate,aluminium acetate, and ammonium acetate. Preferably sodium acetate isused in the kits as described herein. Said acetate salt can be presentin solid form or can be comprised in a buffered solution or buffer.

The amount of salt of the acetate present in the kit as described hereincan be adapted according to the type and/or the kind of gallium-68generator, in particular the quantity of acetate salt present in the kitis able to balance the pH, i.e. to manage the quantity of HCl as elutedfrom a gallium-68 generator such that the resulting solution has a pHbetween 3 and 5, preferably between 3.5 and 4.7, preferably between 3.9and 4.5.

Alternatively, the kit as described in the present invention maycomprise a fixed quantity of acetate salt. The amount of HCl differencesfrom the generator eluate (depending on the type and/or the generatorbrand gallium-68) can then be adjusted by adding an appropriate amountof HCl to the kit as described herein prior to elution. The amount ofHCl added to the kit as described in the present invention is partiallyneutralizing the acetate salt such that the non-neutralized acetate saltis able to balance the pH of a quantity of HCl from a generator eluatesuch that the resulting solution has a pH between 3 and 5, preferablybetween 3.5 and 4.7, preferably between 3.9 and 4.5.

Preferably, the acetate salt is present in the kit as taught herein inan amount between about 1 mg and about 1000 mg, preferably in an amountbetween about 10 mg and about 750 mg, more preferably in an amountbetween about 20 mg and about 500 mg.

Metal inhibitors used in the present invention are selected for theirability to inhibit the competing metals, without (substantially)inhibiting gallium-68 ions in their chelation reaction with thechelate-functionalized targeting agent. Indeed, these metal inhibitorsshould (substantially) not interfere negatively on the mainradiolabelling reaction or lead to the formation of secondaryradiolabeled species. In other words metal inhibitors should have alimited or no capacity to complex gallium-68 in the conditions used forthe radiolabelling reaction, i.e. below 50° C. in an acetate bufferbetween pH 3 and pH 5. Limited means at least 100 times less than thechelating agent used for the chelate-functionalized targeting agent.

It is interesting to note that the function of metal inhibitors in thepresent invention is the opposite of the function of the sequesteringagents used in the prior art. Indeed, according to known methods, at theend of the labelling reaction, a sequestering agent having a particularaffinity for the gallium-68 may be added to chelate the unreactedportion of the isotope, whereas, according to the present invention anagent capable of reducing the competition of metallic impurities otherthan the gallium-68 is added at the beginning of the reaction.

In addition, being able to perform the radiolabelling reaction at atemperature close to room temperature (<50° C.) advantageously allowsthe use of metal inhibitors that would not be usable at the usualtemperatures of radiolabelling DOTA-functionalized targeting agents bysuch as used in WO2013024013, because they would be entering in directcompetition with gallium-68 at such temperatures of above 50° C. Thetemperature is therefore also described in the invention as a parameterfor adjusting the reactivity of the metal inhibitor.

As used herein, a “metal inhibitor” refers to any molecule capable ofinteracting with, or competing metals, or the chelating moiety of thechelate-functionalized targeting agent or with gallium-68 directly, toinhibit wholly or partially the chelation the chelate-functionalizedtargeting agent said competing metals and/or promote the chelating ofgallium-68 by said targeting agent. Such metal inhibitors should have alimited or no capacity to complex gallium-68 in the conditions used forthe radiolabelling reaction, i.e. below 50° C. in an acetate bufferbetween pH 3 and pH 5. Limited means at least 100 times less than thechelating agent used for the chelate-functionalized targeting agent.

Metal inhibitors are preferably selected from the group comprising orconsisting of: DOTA and its derivatives, such as, DOTATOC, DOTANOC,DOTATA, TRITA, DO3A-Nprop, BisDO3A and TrisDO3A; DTPA and itsderivatives such as tetra-tBu-DTPA, p-SCN-Bz-DTPA, MX-DTPA and CHX-DTPA;and sugars. Sugars used as metal inhibitors in the kit of the inventioncan be monosaccharides or derivatives of monosaccharides such astetracetose, pentacetose, hexacétose, tetrose, pentose, hexose,D-mannose, D-fructose, and derivatives; and/or disaccharides and theirderivatives such as maltose and its derivatives; and/or polysaccharidesand their derivatives such as dextrins, cyclodextrins, cellulose andderivatives thereof.

Preferably, the metal inhibitor is present in the kit as describedherein in micromolar amounts, preferably in nanomolar quantities,preferably in an amount of less than 500 nanomolar, still morepreferably in an amount less than 100 nanomoles.

It is important to note that metal inhibitors as shown above can also beadvantageously used in chelation reactions wherein other buffers thanbuffered acetic acid/acetate are used.

Metal inhibitors as shown above can also be advantageously used inchelation reactions wherein said metal inhibitor is included in theeluent generator, in the HCl solution, or in water possibly added beforeelution of the generator. Said metal inhibitor is thus found in theradiolabelling solution. The metal inhibitor may also be chemicallybound to the chelate-functionalized targeting agent. This chemical bondcan or cannot be a labile bond under the conditions of radiolabellingwith the chelate-functionalized targeting agent. This means that in theconditions of radiolabelling the metal inhibitor is formed and releasedin situ. Examples of such preferred bonds are . . .

As used herein, a “chelate-functionalized targeting agent” refers to atargeting agent capable of being labeled with a radioisotope such as forexample gallium-68, by means of a chelating agent which is bound to thetargeting molecule.

Preferred chelating agents for functionalizing a targeting agent to beradiolabeled with gallium-68 are those which form stable chelates withGa3+, in particular 68-Ga3+ (the radioisotope generator eluted from agermanium-68/gallium-68 generator using HCl), at least for a timesufficient for diagnostic investigations using such radiolabelledtargeting agents. Suitable chelating agents include aliphatic amines,linear or macrocyclic such as macrocyclic amines with tertiary amines.While these examples of suitable chelating agents are not limited, theypreferably include the NOTA and its derivatives, such as TACN, TACN-TM,DTAC, H3NOKA, NODASA, NODAGA, NOTP, NOTPME, PrP9, TRAP, Trappist Pr,NOPO, TETA; Tris(hydroxypyridinone) (THP) and derivatives, chelates openchain such as HBED, DFO or desferrioxamine or desferal, EDTA, 6SS, B6SS,PLED, TAME, YM103; NTP (PRHP) 3; the H2dedpa and its derivatives such asH2dedpa-1, 2-H2dedpa, H2dp-bb-NCS, and H2dp-N-NCS; (4,6-MeO2sal)2-BAPEN; and citrate and derivatives thereof.

The chelate-functionalized targeting agent can be a peptide, forexample, a peptide comprising 2 to 20 amino acids, a polypeptide, aprotein, a vitamin, a saccharide, for example a monosaccharide or apolysaccharide, an antibody and its derivatives such as nanobodies,diabodies, antibodies fragments, nucleic acid, an aptamer, an antisenseoligonucleotide, an organic molecule, or any other biomolecule that isable to bind to a certain diagnostic target or to express a certainmetabolic activity.

Chelate-functionalized targeting agents as described herein preferablyhave a capacity of biological targeting. Non-limiting examples ofsuitable targeting agents include molecules that target VEGF receptors,analogs of bombesin or GRP receptor targeting molecules, moleculestargeting somatostatin receptors, RGD peptides or molecules targetingαvβ3 and αvβ5, annexin V or molecules targeting the apoptotic process,molecules targeting estrogen receptors, biomolecules targeting theplaque . . . More generally, a list targeting molecules, organic or not,functionalized by a chelating agent can be found in the journal ofVelikyan et al., Theranostic 2014, Vol. 4, Issue 1 “Prospective of68Ga-Radiopharmaceutical Development.”

In some embodiments, the metal inhibitor is included in the eluentgenerator, in the HCl solution, or possibly in the added water prior toelution of the generator. Said metal inhibitor and is thus found in theradiolabelling solution.

The various components of the kit as described herein are preferablypresent in a container or vial, preferably a siliconized glass vial.However, also a kit wherein the individual components are present inseparate containers or vials is envisaged.

The invention further provides a method for radiolabelling a targetingagent with gallium-68, said method comprising the elution of agallium-68 generator with an eluent comprising an acid, in a kit asdescribed herein, e.g. comprising the metal inhibitor, thechelate-functionalized targeting agent and acetate salt.

As indicated above, when the chelate-functionalized targeting agent isincluded in the kit, a gallium-68 generator can be eluted directly intothe kit. In other embodiments, the chelate-functionalized targetingagent can be added to a kit comprising the acetate salt and ametal-inhibiting agent as described herein, prior to elution.

In some embodiments, the gallium-68 generator is eluted directly intothe kit. In other embodiments, water is added to the solution prior toelution.

In some embodiments of the present invention, an appropriate amount ofHCl is added to the solution prior to elution. Said HCl is added topartially neutralize the acetate. The amount of HCl added, preferablypartially neutralizes the quantity of acetate salt in such a manner thatthe remaining quantity of acetate salt, i.e. unneutralized acetate salt,is able to balance the pH of said amount of HCl from the generatoreluate (and thus dedicated to one type or brand of given generator) suchthat the pH of the solution obtained for the radiolabelling reaction orchelating reaction, resulting from the addition of HCl and the generatoreluate in the kit as described herein, is in a pH range between 3 and 5,preferably between 3.5 and 4.5, preferably between 3.9 and 4.3. Said HClmay be added directly to the solution, or after a certain amount ofwater is added to said kit.

All gallium-68 generator may be used in the methods of the presentinvention. Typically, a commercial gallium-68 generator comprises acolumn on which the germanium-68 is fixed. A gallium-68 generator istypically eluted with an eluent comprising an acid, preferably HCl.Therefore, in preferred embodiments of the method, as taught herein, thegallium-68 generator is eluted with an eluent comprising HCl.

After elution of the gallium-68 generator in the kit as describedherein, the solution obtained is left to react in the radiolabellingreaction for a short period of time, in particular between about 2minutes and about 60 minutes, preferably from about 2 minutes to about30 minutes, for example about 10 minutes.

Preferably, the radiolabelling reaction or chelation is performed at atemperature below 50° C., preferably of below 45° C., below 40° C.,below 35° C., or below 30° C., most preferably at room temperature, e.g.between 20 and 25° C.

Preferably, the radiolabelling reaction or chelation is performed at apH between about 3 and about 5, more preferably between about 3.5 andabout 4.5, more preferably between about 3.9 and about 4.3.

The invention also encompasses the solution obtained by elution of agallium-68 generator with an eluent comprising an acid, preferably HCl,in a kit as taught herein.

Preferably, said solution has a pH between about 3 and about 5,preferably between about 3.5 and about 4.5, more preferably betweenabout 3.9 and about 4.3.

The invention also discloses a gallium-68 radiolabeled targeting agent,obtained by anyone of the methods as described herein.

In one aspect, the invention also provides a preparation method of a kitas described herein, said method comprising the steps of:

a) preparing a solution comprising the acetate salt, achelate-functionalized targeting agent and an inhibitor of metal; and

b) lyophilizing the solution obtained in step a).

Alternatively, the invention further provides a process for preparing akit of the invention comprising the steps of:

a) preparing a solution comprising a chelate-functionalized targetingagent and an inhibitor of metal; and

b) lyophilizing the solution obtained in step a).

c) adding the acetate salt as a powder in the obtained lyophilizedproduct in step b).

Further alternatively, the invention further provides a process forpreparing a kit of the invention comprising the steps of:

a) preparing a solution comprising a chelate-functionalized targetingagent and an inhibitor of metal; and

b) lyophilizing the solution obtained in step a).

c) adding an acetate buffer in the obtained lyophilized product in stepb).

Finally, the invention further provides a process for preparing a kit ofthe invention comprising the steps of:

a) preparing a solution comprising the acetate salt, achelate-functionalized targeting agent and an inhibitor of metal; and

b) optionally freeze the solution obtained in step a).

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing description. Accordingly, it is intendedto embrace all such alternatives, modifications, and variations asfollows in the spirit and broad scope of the appended claims.

The above aspects and embodiments are further supported by the followingnon-limiting examples.

EXAMPLES Example 1: Generator E & Z/NODAGA Peptide without MetalInhibitor

Labelling a Peptide with a 68Ga Eluate of 5 mL of 0.1 M HCl

A commercial gallium-68 generator 1850 MBq (Eckert & Ziegler) is elutedwith 5 mL of 0.1M HCl (Ultrapure grade) directly into a flask containing150 mg of sodium acetate (Ultrapure grade) lyophilized, 240 μl of HCl 3M(Ultrapure grade), 760 μl of Milli-Q and 50 μg lyophilized NODAGA-NOC.The flask was left for 10 min at room temperature. The product isobtained with a radiochemical purity of 64% according to TLC analysis ofthe reaction medium.

Example 2: Similarly to What was Done in Example 1 DifferentCombinations were Tested and are Summarized in the Table Below

Radiolabelling yield for 10 minutes, room Chelating agent T° vsradiolabelling use in the chelate-  yield without metal functionalizedinhibitor using Generator Kit Entry Acetate targeting agent InhibitorGenerator similar conditions cleaniliness Preparation* 1 150 mg NOTA 25μg DOTA E&Z 82% vs 51% Generator A cleaned 2 150 mg NOTA 25 μg FructoseE&Z 87% vs 51% Generator A cleaned 3 150 mg NOTA 25 μg Beta- E&Z 83% vs51% Generator A cyclodextrin cleaned 4 150 mg NODAGA 25 μg Beta- E&Z 95%vs 64% Generator A cyclodextrin cleaned 5 150 mg HBED 25 μg Beta- E&Z91% vs 77% Generator A cyclodextrin cleaned 6 150 mg HBED 25 μg FructoseE&Z 94% vs 77% Generator A cleaned 7 150 mg NOTA 10 μg Fructose E&Z 85%vs 39% Generator A cleaned 9 150 mg NODAGA 10 μg Beta- E&Z 84% vs 55%Generator A cyclodextrin cleaned 9 150 mg HBED 10 μg Beta- E&Z 87% vs51% Generator A cyclodextrin cleaned 10 150 mg NODAGA 50 μg Beta- ITG94% vs 46% Generator A cyclodextrin cleaned 11 150 mg NODAGA 50 μg Beta-E&Z 97% vs 70% Generator A cyclodextrin cleaned 12 150 mg NODAGA 50 μgD-Mannose E&Z 91% vs 44% Generator A not cleaned 13 150 mg NODAGA 50 μgDOTA E&Z 95% vs 70% Generator A cleaned 14 150 mg NODAGA 50 μg Beta-iThemba 91% vs 61% Generator A cyclodextrin cleaned 15 150 mg NODAGA 50μg Fructose E&Z 95% vs 70% Generator A cleaned 16 150 mg HBED 20 μg DOTAITG 91% vs 75% Generator A cleaned 17 150 mg NODAGA 25 μg D-Mannose ITG95% vs 60% Generator A cleaned 18 150 mg NODAGA 25 μg Beta- ITG 96% vs60% Generator A cyclodextrin cleaned 19 150 mg NODAGA 25 μg tetra-tBu-ITG 89% vs 60% Generator A DTPA cleaned 20 150 mg NODAGA 25 μg Beta- ITG96% vs 61% Generator B cyclodextrin cleaned 21 150 mg NODAGA 25 μg DOTAE&Z 94% vs 64% Generator B cleaned 22 150 mg NODAGA 25 μg DOTA E&Z 89%vs 64% Generator C cleaned 23 150 mg NODAGA 25 μg Glucose ITG 89% vs 61%Generator C cleaned 24 150 mg DFO 10 μg DOTA ITG 98% vs 85% Generator Acleaned * A = a preparation method comprising the steps of: a) preparinga solution comprising the acetate salt, a chelate-functionalizedtargeting agent and an inhibitor of metal; and b) lyophilizing thesolution obtained in step a). B = a preparation method comprising thesteps of: a) preparing a solution comprising a chelate-functionalizedtargeting agent and an inhibitor of metal; and b) lyophilizing thesolution obtained in step a). c) adding the acetate salt as a solid C =a preparation method comprising the steps of: a) preparing a solutioncomprising a chelate-functionalized targeting agent and an inhibitor ofmetal; andb) lyophilizing the solution obtained in step a). c) addingthe acetate salt as a buffer solution adapted to the generator used

To conclude, the results above clearly show the increased gallium-68radiolabelling yield of about 90% or more in all set-ups where a metalinhibitor as defined herein is used in addition to thechelator-functionalized targeting agent. If said agent is not added,much lower yields are obtained. The yield is virtually independent ofthe use of acetate in solid form or in buffer form. Also when theacetate salt is co-lyophilized with the metal inhibitor and thechelator-functionalized targeting agent, a very good yield is obtained.

1-29. (canceled)
 30. A radiolabelling kit comprising: an acetate salt orbuffer in an amount sufficient to balance at least the acidic pH eluatefrom a gallium-68 generator to a pH value ranging from 3 to 5 when saidgenerator is eluted in the kit; and an HBED-functionalized targetingagent, said HBED-functionalized targeting agent being able to chelategallium-68 in radiolabelling conditions, wherein said acetate salt andHBED-functionalized targeting agent are co-lyophilized.
 31. The kitaccording to claim 30, wherein additionally a metal inhibitor is presentselected from the group consisting of DOTA and its derivatives, DTPA andits derivatives, and sugars, wherein said metal inhibitor and saidHBED-functionalized targeting agent are not chemically linked.
 32. Thekit according to claim 31, wherein said metal inhibitor is selected fromthe group consisting of monosaccharides and disaccharides.
 33. The kitaccording to claim 31, wherein said metal inhibitor is co-lyophilizedwith the acetate buffer and HBED-functionalized targeting agent.
 34. Thekit according to claim 30, wherein said HBED-functionalized targetingagent is PSMA-11.
 35. A method for radiolabelling an HBED-functionalizedtargeting agent with gallium-68, comprising the elution of a gallium-68generator with an eluent comprising an acid, in a kit according to claim30 and allowing the HBED-functionalized targeting agent to be labelledwith gallium-68.
 36. The method according to claim 35, wherein the acidis HCl.
 37. The method according to claim 35, wherein the radiolabellingis performed at a pH between 3 and
 5. 38. The method according to claim35, wherein the radiolabeling reaction is carried out at ambient or roomtemperature.
 39. An in vivo method of imaging in a subject comprisingthe steps of: 1) producing a gallium-68 radiolabeled HBED-functionalizedtargeting agent using the method according to claim 35; 2) administeringsaid gallium-68 radiolabeled HBED-functionalized targeting agent to thesubject; and 3) detecting the said gallium-68 radiolabeledHBED-functionalized targeting agent in the subject using positronemission tomography (PET).
 40. The method according to claim 39, whereinsaid HBED-functionalized targeting agent is PSMA-11.
 41. The methodaccording to claim 35, wherein the radiolabelling is performed at a pHbetween 3.5 and 4.5.
 42. The method according to claim 35, wherein theradiolabelling is performed at a pH between 3.9 and 4.3.
 43. The methodaccording to claim 35, wherein the radiolabeling reaction is carried outat a temperature between 20 and 30° C.